Human G-CSF belongs to hematopoetic growth factors which have a decisive role in the formation of neutrophils. G-CSF is used in medicine in the field of hematology and oncology. Today, two forms of G-CSF for clinical use are in the market: lenograstim which is glycosylated and is produced in mammalian cells, specifically a CHO cell line (Holloway C J (1994) Eur J Cancer 30A Suppl 3:S2-S6; EP 169566), and filgrastim which is non-glycosylated and is produced in a bacterium E. coli (EP 237545).
It is known from the literature that especially non-glycosylated forms of G-CSF are particularly unstable in vitro compared to glycosylated form of G-CSF which, is obtained from CHO cells (Oh-eda et al (1990) J Biol Chem 265(20):11432-35). Due to different stabilities of glycosylated and non-glycosylated G-CSF the prior art approaches for preparation of stable pharmaceutically acceptable G-CSF differ according to the presence/absence of glycosylation. Especially in the case of non-glycosylated G-CSF which is a hydrophobic protein a preparation of stable pharmaceutically acceptable compositions that would support stability of the pharmaceutical proteins on longer storage periods and upon medical application is a difficult task, represents a special challenge and requires specially selected measures.
Stabilized compositions of non-glycosylated G-CSF are described in EP 373679 and are characterized essentially by advantageously providing G-CSF stability at low conductivity and at an acidic pH value ranging from 2.75 to 4.0. However, various sugars or sugar alcohols, amino acids, polymers and detergents can be added for better G-CSF stability. In particular, it is stressed that pH of the composition should be less than 4 in order to reduce aggregate formation and to increase the G-CSF stability. The aggregate formation and the reduced stability of pharmaceutical compositions comprising G-CSF at pH above 4.0 described in EP 373679 are in accordance with the results obtained from prior art literature (Kuzniar et al (2001) Pharm Dev Technol 6(3):441-7; Bartkowski et al (2002) J Protein Chem 21(3):137-43; Narhi et al (1991) J Protein Chem 10(4): 359-367; Wang W (1999) Int J Pharmaceut 185:129-188.
Different approaches of stabilizing G-CSF are described in other patent and scientific literature. EP 1129720 discloses a stable aqueous composition of non-glycosylated bovine G-CSF that has pH in the range from 5 to 8 and comprises a salt comprising sulfate ions at the concentrations of from about 0.01 M to about 1.0 M.
In EP 607156 a pharmaceutical composition containing G-CSF for infusion and injection purposes is disclosed. The stabilisation of G-CSF is achieved by setting an acidic pH value which is favourable for G-CSF and by adding a mixture of various preservatives, different amino acids and a surfactant as stabilizers. It is not clear from the description whether glycosylated or non-glycosylated form of G-CSF was used. EP 674525 discloses aqueous pharmaceutical compositions of G-CSF stabilized in a buffer selected from the group consisting of a salt of citrate, maleate, a combination of phosphate and citrate, and arginine. In addition, at least one surfactant is added for achieving the G-CSF stability. It is not specified in the description whether either glycosylated or non-glycosylated form of G-CSF was used.
GB 2 193621 discloses a composition of glycosylated G-CSF with pH in the range from 7 to 7.5 which comprises at least one substance selected from the group consisting of a pharmaceutically acceptable surfactant, saccharide, protein and a high-molecular weight compound. Suitable high-molecular weight compounds include hydroxypropyl cellulose, hydroxymethyl cellulose, sodium carboxymethyl cellulose, polyethylene glycol, polyvinyl alcohol, and polyvinylpyrrollidone. In particular, compositions are stated to be advantageous which contain a surfactant.
In EP 1060746 a pharmaceutical composition comprising glycosylated G-CSF having pH about 6.5 is described in the presence of at least one pharmaceutically acceptable surfactant. The specification discloses the measurement of stability of desialylated G-CSF depending on pH. The results show a very low stability of desialyated G-CSF at pH in the range from about 5.0 to about 7.0.
EP 0988861 discloses a pharmaceutical composition of G-CSF, especially bovine G-CSF, where the G-CSF stability was achieved by addition of a stabilizing buffer, such as HEPES, TRIS, or TRICINE at the concentrations of about 1 M.
The lyophilised pharmaceutical compositions of G-CSF stabilized by the addition of maltose, cellobiose, gentiobiose, isomaltose, raffinose, trehalose or amino sugar are described in EP 0674524.
Glycosylated G-CSF compositions containing one or more amino acids at pH about 6.5 are described in EP 1197221, EP 1260230 and in EP 1329224, whereas the lyophilised compositions stabilized with amino-acids are described in EP 0975335. The method for prolonging the duration of pharmacological effect of G-CSF characterized in that a surfactant is present in a G-CSF water-soluble solution is described in JP2002371009.
Oral dosage form of G-CSF described in EP 0459795 is based on the stabilization with surfactants, fatty acids and enteric material. The compositions stabilized over long time with the addition of methionine are described in WO51629.
Although low ionic strength is preferential for pharmaceutical compositions of G-CSF, almost in every case in the patent and scientific literature various surfactants are added, either non-ionic, anionic or natural surfactants for prevention of G-CSF aggregation and denaturation at packing material surfaces. However, the surfactants in the G-CSF composition should preferably be avoided from a medical point of view since they can cause local irritations, they may contain toxic impurities which have to be controlled very carefully and additionally it is not always clear how they are metabolized and excreted. In addition, certain surfactants susceptible to autooxydation can damage proteins.
The object of the present invention is to provide a stable liquid pharmaceutical composition of non-glycosylated G-CSF which may enable a proper use of G-CSF as a pharmaceutical agent without the addition of surfactants and additives derived from human and/or animal origin. In particular, the pharmaceutical composition of the present invention has a long shelf life, is physiologically well-tolerated, simple to use and it is possible to dose it precisely.